Mutation and selective constraint in the murid genome

A large proportion of the genome of many higher eukaryotes consists of apparently functionless noncoding DNA, the significance of which is a long­standing puzzle in biology. The aim of this work was to quantify the extent to which both mutation and natural selection have influenced molecular evolution in murid noncoding sequence. In particular, the magnitude of and variation in selective constraint within murid noncoding DNA was investigated. Selective constraint is defined as the proportion of all mutations occurring at a locus or site which are strongly deleterious and therefore removed by natural selection. The approach adopted to estimate selective constraint relies on the assumption that we can quantify the past strength of purifying selection in a DNA sequence by comparison with nearby regions which are assumed to be evolving neutrally. To this end, work in this thesis deals both with mutational variation and bias (Chapters 2 and 3) as well as with selective constraint (Chapters 4 and 5) in noncoding DNA.Chapter 2 is concerned with the differential effects of context-dependent mutation (namely, CpG hypermutability) at fourfold synonymous and noncoding sites. Using simulations it was shown that a common method of assigning ancestral CpG status often introduces a substantial level of bias into the estimation of nucleotide substitution rates. The effects of this bias can easily be misconstrued as the action of purifying selection at synonymous sites.Chapter 3 is concerned with mutational variation in the murid genome. Nucleotide substitution rates in murid transposable elements were estimated. It was assumed that the majority of murid transposable elements were evolving neutrally and, therefore, that their molecular evolutionary rate was dictated by mutation alone. Under this assumption, variation in estimated element substitution rates reflects sampling and mutational variation only. The results indicate that greater mutational variation occurs along the length of a chromosome than between individual chromosomes, although the latter has been the primary focus in the literature. This result illustrates the importance of accounting for mutational variation in studies of selective constraint and sequence conservation.In Chapter 4, the level of constraint in intergenic DNA adjacent to coding sequences and a moderate distance inside first introns was estimated in a sample of 300 mouse-rat gene orthologues. The results suggested that whilst selective constraint in intergenic sequence adjacent to the start and stop codons is moderately high, this becomes statistically indistinguishable from zero within 4kb upstream/downstream of the first/last exon. Selective constraint in the 5' end of the first intron was also found to be moderately high. Taking the contributions from noncoding sequence into account, it was estimated that the number of deleterious mutations occurring in murid noncoding DNA was approximately equal to that in protein-coding sequence.Chapter 5 expands on the work done in Chapter 4. The assumption of neutral evolution in non-first introns was addressed by comparing their evolutionary rates with those in transposable elements. In addition the selective constraint in intergenic DNA immediately adjacent to genes with that found large distances from known genes was compared. The results showed that, when repetitive sequence is removed, the selective constraints in intergenic DNA are significantly different from zero. Furthermore, this constraint does not become indistinguishable from zero, even at large distances ( 50kb) from genic regions. The data also showed that a weak correlation between intron length and nucleotide substitution rate exists in murid non-first introns.

Effect of the assignment of ancestral CpG state on the estimation of nucleotide substitution rates in mammals

<p>Abstract</p> <p>Background</p> <p>Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related.</p> <p>Results</p> <p>Using simulations of DNA sequence evolution we show that assignment of the ancestral CpG state based on the simple presence/absence of the CpG dinucleotide can seriously bias estimates of the substitution rate, because many true non-CpG changes are misassigned as CpG. Paradoxically, this bias is most severe between closely related species, because a minimum of two substitutions are required to misassign a true ancestral CpG site as non-CpG whereas only a single substitution is required to misassign a true ancestral non-CpG site as CpG in a two branch tree. We also show that CpG misassignment bias differentially affects fourfold degenerate and noncoding sites due to differences in base composition such that fourfold degenerate sites can appear to be evolving more slowly than noncoding sites. We demonstrate that the effects predicted by our simulations occur in a real evolutionary setting by comparing substitution rates estimated from human-chimp coding and intronic sequence using CpG/non-CpG assignment with estimates derived from a method that is largely free from bias.</p> <p>Conclusion</p> <p>Our study demonstrates that a common method of assigning sites into CpG and non CpG classes in pairwise alignments is seriously biased and recommends against the adoption of <it>ad hoc </it>methods of ancestral state assignment.</p

Cell reprogramming shapes the mitochondrial DNA landscape.

Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10-5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy

Alternative splicing is frequent during early embryonic development in mouse

<p>Abstract</p> <p>Background</p> <p>Alternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development.</p> <p>Results</p> <p>A genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, <it>Fox2</it>, we demonstrate that changes in <it>Fox2 </it>expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites.</p> <p>Conclusions</p> <p>We propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies</p

Peak event: the rise, crisis and potential decline of the Olympic Games and the World Cup

This paper tracks the growth of two of the largest tourist events: the Olympic Games and the Football World Cup, drawing on a dataset containing all events between 1964 and 2018. Overall, the size of the three events has grown about 60-fold over the past 50 years, thirteen times faster than world GDP. We identify an S-shaped growth curve and four different growth periods, with an emergent crisis phase in the late 2010s that may have brought us to ‘peak event’ – the point at which these events have reached their largest size. Outlining three different scenarios, we argue that the Olympics and the World Cup are at a critical bifurcation point, which also requires new bidding and hosting policies

Spatially and Temporally Resolved Measurements of Velocity in a H2-air Combustion-Heated Supersonic Jet

This paper presents simultaneous measurements at multiple points of two orthogonal components of flow velocity using a single-shot interferometric Rayleigh scattering (IRS) technique. The measurements are performed on a large-scale Mach 1.6 (Mach 5.5 enthalpy) H2-air combustion jet during the 2007 test campaign in the Direct Connect Supersonic Combustion Test facility at NASA Langley Research Center. The measurements are performed simultaneously with CARS (Coherent Anti-stokes Raman Spectroscopy) using a combined CARS-IRS instrument with a common path 9-nanosecond pulsed, injection-seeded, 532-nm Nd:YAG laser probe pulse. The paper summarizes the measurements of velocities along the core of the vitiated air flow as well as two radial profiles. The average velocity measurement near the centerline at the closest point from the nozzle exit compares favorably with the CFD calculations using the VULCAN code. Further downstream, the measured axial velocity shows overall higher values than predicted with a trend of convergence at further distances. Larger discrepancies are shown in the radial profiles